EP1434596B1 - Amelioration des reponses immunitaires par des anticorps agonistes liant 4-1bb - Google Patents

Amelioration des reponses immunitaires par des anticorps agonistes liant 4-1bb Download PDF

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EP1434596B1
EP1434596B1 EP02802551A EP02802551A EP1434596B1 EP 1434596 B1 EP1434596 B1 EP 1434596B1 EP 02802551 A EP02802551 A EP 02802551A EP 02802551 A EP02802551 A EP 02802551A EP 1434596 B1 EP1434596 B1 EP 1434596B1
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cell
antigen
cells
peptide
antibody
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Lieping Chen
Scott E. Strome
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Mayo Foundation for Medical Education and Research
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Definitions

  • This invention relates to immunoregulation, and more particularly to T cell response regulation.
  • Mammalian T lymphocytes recognize antigenic peptides bound to major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells (APC).
  • MHC major histocompatibility complex
  • APC antigen presenting cells
  • the antigenic peptides are generated by proteolytic degradation of protein antigens within the APC.
  • the interaction of the T cells with the APC and the subsequent response of the T cells are qualitatively and quantitatively regulated by interactions between cell surface receptors on the T cells with both soluble mediators and ligands on the surface of APC.
  • TAKAHASHI C. et al., Journal of Immunology, 1999, Vol. 162, No. 9, pages 5037-5040 disclose the use of an agonist 4-1BB antibody in combination with staphylococcal enterotoxin A (SEA).
  • SEA staphylococcal enterotoxin A
  • peripheral T cells will clonally expand. After expansion, there is a dramatic decrease in the frequency and absolute number of SEA-specific T cells.
  • SEA is injected with an agonist mAb specific for 4-1BB in mice, clonal expansion of the CD4 V ⁇ 3 T cells is significantly increased. Furthermore, the deletion of expanded CD8 T cells was inhibited.
  • mice bearing a weakly immunogenic tumor with an agonistic antibody specific for murine 4-1BB (also known as CD137) and a peptide fragment of a polypeptide expressed by the tumor resulted in regression of the tumor.
  • an agonistic antibody specific for murine 4-1BB also known as CD137
  • treatment of mice bearing a second weakly immunogenic tumor with the same 4-1BB antibody and autologous dendritic cells "primed” in vitro with cells of the tumor resulted in regression of the tumor.
  • signaling via cell-surface 4-1BB molecules and providing an immunogenic stimulus (a) prevents induction of anergy in CD8+ T cells and (b) reverses already established anergy in the CD8+ T cells.
  • the invention features (a) an antigen for which a T cell receptor is specific such as a tumor associated pcptide-epitope and (b) an agonistic 4-1BB-binding antibody) for use as a medicament or a combination of medicaments for generating an enhanced immune response in a subject.
  • the invention also features an in vitro method of enhancing the response of a T cell in which a population of cells containing a T cell is incubated with (a) an antigen for which a T cell receptor is specific such as a peptide-epitope from an infectious microoganism and (b) an agonistic 4-1BB-binding antibody.
  • the invention also embraces an in vitro method of activating a T cell, e.g., a CD8+ T cell or a CD4+ T cell.
  • This method involves: (a) providing a cell sample comprising a T cell; and (b) culturing the cell sample with an antigen for which a T cell receptor is specific and an agonistic 4-1BB-binding antibody.
  • Another aspect of the invention is a method of preventing induction of anergy or of reversing anergy in a T cell; the method includes contacting the T cell with: (a) an antigen for which a T cell receptor is specific; and (b) an agonistic 4.1BB-binding antibody.
  • the contacting can be in vitro .
  • the invention also features (a) an antigen for which a T cell receptor is specific and an agonistic 4-1BB-binding antibody; (b) a nucleic acid encoding the antigen and the agonistic 4-1BB-binding antibody ; (c) the antigen and a nucleic acid encoding the agonistic 4-1BB binding antibody; or (d) a nucleic acid encoding the antigen and a nucleic acid encoding the agonistic 4-1BB binding antibody for use as a medicament or a combination of medicaments for preventing induction of angergy or reversing anergy in a T cell in a subject.
  • nucleic acid encoding the antigen - and a nucleic acid encoding the 4-1BB binding antibody the nucleic acid encoding the antigen and the nucleic acid encoding the 4-1BB binding antibody being in the same nucleic acid molecule.
  • nucleic acid encoding the a ntigen or the 4-1BB-binding agent may be transfected or transduced into a cell, the cell being a cell, or a progeny of a cell, that prior to the transfection or the transduction, was obtained from a mammal.
  • the agonistic 4-1BB binding antibody can be a functional fragment thereof.
  • the antigen can be a (a) a tumor-associated antigen (TAA) or (b) a functional fragment of a TAA and it can be a polypeptide.
  • TAA can be a molecule produced by a leukemia, a lymphoma, a neurological cancer, a melanoma, a breast cancer, a lung cancer, a head and neck cancer, a gastrointestinal cancer, a liver cancer, a pancreatic cancer, a genitourinary cancer, a prostate cancer, a renal cell cancer, a bone cancer, or a vascular cancer cell.
  • the antigen -can be a dendritic cell that has a major histocompatibility complex (MHC) molecule with peptide-epitope bound thereto, the peptide-epitope being a fragment of a TAA or a fragment of a polypeptide produced by an infectious microorganism.
  • MHC major histocompatibility complex
  • the MHC molecule can be a MHC class I molecule or a MHC class II molecule.
  • the antigen -can be also be a hybrid cell, e.g., a fusion product of a tumor cell and a dendritic cell.
  • the antigen can be a tumor cell, a tumor cell lysate, a TAA, a peptide-epitope of a TAA, or a heat shock protein bound to peptide-epitope of protein expressed by a tumor cell.
  • the antigen can also be a dendritic cell that has been incubated with tumor cells, a tumor cell lysate, a TAA, a peptide-epitope of a TAA, or a heat shock protein bound to peptide-epitope of protein expressed by a tumor cell.
  • the cell can be transfected with or transformed with a nucleic acid encoding a cytokine or a growth factor, e.g., granulocyte macrophage-colony stimulating factor (GM-CSF).
  • a cytokine or a growth factor, e.g., granulocyte macrophage-colony stimulating factor (GM-CSF).
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • the antigen can be a molecule produced by an infectious microorganism, e.g., a virus such as a retrovirus, a bacterium, a fungus, or a protozoan parasite.
  • an infectious microorganism e.g., a virus such as a retrovirus, a bacterium, a fungus, or a protozoan parasite.
  • an "enhanced immune response” is obtained by administering to a subject antigen for which a T cell receptor is specitic and an agonistic 4-1BB-binding antibody.
  • an appropriate antigen either stimulates no immune response in the subject or it stimulates an immune response in the subject that is detectably lower than a response stimulated by administration of the antigen and an agonistic 4-1BB-binding agent.
  • an "anergic T cell” or an “anergized T cell” is a T cell whose ability to respond to an immunogenic stimulus, with respect to at least one activity of the T cell, has been partially or completely inhibited.
  • an anergic or anergized T cell may lack or have a decreased ability to proliferate and produce interleukin-2 in response to an immunogenic stimulus but its ability to produce interferon- ⁇ in response to the immunogenic stimulus may be substantially intact.
  • an anergic or anergized T cell may display a lack or a decrease in all the functional activities elicited by an immunogenic stimulus in such a T cell.
  • reversing anergy in a T cell means fully or partially restoring the ability of the T cell to perform one or more functions in response to an immunogenic stimulus.
  • preventing the induction of" anergy in a T cell means fully or partially inhibiting the induction of anergy in the T cell.
  • an "agonistic 4-1BB-binding agent” is a substance that upon binding to a 4-1BB molecule on a target cell (e.g., a T cell) enhances the response of the target cell to an immunogenic stimulus.
  • a "functional fragment of a tumor-associated antigen (TAA)” is a fragment of a TAA shorter than the full-length TAA but with greater than 10% (e.g., greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 98%, greater than 99%, greater than 99.5%, or 100% or more) of the ability of the full-length TAA to activate an immune response in the presence or absence of an agonistic 4-1BB-binding agent.
  • TAA that are polypeptides a "full-length" TAA is the mature TAA, i.e.. the polypeptide lacking its native signal sequence.
  • a "peptide-epitope" of a polypeptide is a fragment of a polypeptide that binds to a major histocompatibility complex (MHC) molecule and is recognized in the form of a complex with the MHC molecule by an antigen specific receptor on a T cell (TCR).
  • MHC molecules can be class I or class II MHC molecules.
  • Polypeptide and “protein” are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification.
  • the invention is based on the finding that treatment of mice bearing a weakly immunogenic tumor with an agonistic antibody specific for murine 4-1BB and a peptide fragment of a polypeptide expressed by the tumor resulted in regression of the tumor.
  • treatment of mice bearing a second weakly immunogenic tumo r with the same 4-1BB antibody and autologous dendritic cells "primed" in vitro with cells of the tumor resulted in regression of the tumor.
  • the inventors have also discovered that providing an immunogenic stimulus and a 4-1BB-mediated signal to a CD8+ T cell not only prevents induction of anergy in a CD8+ T cell, but can also reverse, partially or completely, already established anergy in a CD8+ T cell.
  • the invention features methods of activating mammalian immune responses in which cells of the immune system are exposed to (a) an antigen for which a T cell receptor is specific and (b) an agonistic 4-1BB-binding antibody. Exposure of the cells to the antigen can occur before, during, or after exposure to the 4-1BB binding antibody. The two exposures will preferably be substantially simultaneous.
  • Responses that are enhanced by the methods of the invention can be any immune response.
  • the responses enhanced are preferably T cell responses.
  • enhancement of a CD4+ T cell helper cell response can indirectly result in enhancement of a B cell antibody response.
  • the activities of other cells of the immune system e.g., monocytes/macrophages, granulocytes (e.g., neutrophils), and natural killer cells
  • monocytes/macrophages, granulocytes (e.g., neutrophils) are regulated by T cells.
  • the methods of the invention can be used to enhance responses of any or all of these cell types.
  • the invention also provides methods for preventing induction of anergy in r cells or reversing anergy in T cells already rendered anergic.
  • the relevant T cells are contacted with an antigen for which a T cell receptor is specific and a 4-1BB-binding antibody. Contacting of the T cells with the antigen can occur before, during, or after contacting the T cells with the 4-1BB binding agent.
  • an "immunogenic stimulus” is a stimulus delivered to a T cell via the antigen-specific T cell receptor (TCR) expressed on the surface of the T cell More commonly, but not necessarily, such a stimulus is provided in the form of an antigen for which the TCR is specific. While such antigens will generally be protein, they can also be carbohydrates, lipids, nucleic acids or hybrid molecules having components of two or more of these molecule types, e.g., glycoproteins or lipoproteins. However, the immunogenic stimulus can also be provided by other agonistic TCR ligands such as antibodies specific for TCR components (e.g., TCR ⁇ -chain or ⁇ -chain variable regions) or antibodies specific for the TCR-associated CD3 complex.
  • TCR antigen-specific T cell receptor
  • Immunogenic stimuli do not include antigen-non-specific stimuli provided by, for example, cytokines (e.g., interleukin-12), growth factors, co-stimulatory molecules, or adhesion molecules. While such stimuli can be exploited in the methods of the invention, they do not constitute the required immunogenic stimulus.
  • Antigens useful as immunogenic stimuli include alloantigens (e.g., a MHC alloantigen) on, for example, an antigen presenting cell (APC) (e.g., a dendritic cell (DC), a macrophage, a monocyte, or a B cell).
  • APC antigen presenting cell
  • DC dendritic cell
  • macrophage a monocyte, or a B cell
  • DC of interest are interdigitating DC and not follicular DC; follicular DC present antigen to B cells.
  • interdigitating DC are referred to herein as DC.
  • Methods of isolating DC from tissues such as blood, bone marrow, spleen, or lymph node are known in the art, as are methods of generating them in vitro from precursor cells in such tissues.
  • Also useful as immunogenic stimuli are polypeptide antigens and peptide-epitopes derived from them. Unprocessed polypeptides are processed by APC into peptide-epitopes that are presented to responsive T cells in the form of molecular completes with MHC molecules on the surface of the APC.
  • Useful immunogenic stimuli also include a source of antigen such as a lysate of either tumor cells or cells infected with an infectious microorganism of interest.
  • APC e.g., DC
  • pre-exposcd e.g., by coculturing
  • antigenic polypeptides, peptidc-epitopes of such polypeptides or lysates of tumor (or infected cells) can also be used as immunogenic stimuli.
  • Such APC can also be "primed” with antigen by culture with a cancer cell or infected cell of interest; the cancer or infected cells can optionally be irradiated or heated (e.g., boiled) prior to the priming culture.
  • APC especially DC
  • antigen as an immunogenic stimulus can be provided in the form of cells (e.g., tumor cells or infected cells producing the antigen of interest).
  • immunogenic stimuli can be provided in the form of cell hybrids formed by fusing APC (e.g., DC) with tumor cells [ Gong et al. (2000) Proc. Natl. Acad. Sci. USA 97(6):2716-2718 ; Gong et al. (1997) Nature Medicine 3(5):558-561 ; Gong et al. (2000) J. Immunol. 165(3):1705-1711 ] or infected cells of interest.
  • APC e.g., DC
  • IC immunogenic cells
  • Cells or cell hybrids can be used (as immunogenic stimuli) untreated or they can be metabolically inhibited (e.g., by irradiation or exposure to a drug such as mitomycin-C) so as to substantially ablate their ability to divide.
  • Tumor or infected cells used per se as an immunogenic stimulus or as IC for the production of cell hybrids will preferably, but not necessarily, be derived from the same donor as that of the T cell.
  • the cell s are from a different donor, they will preferably share one MHC haplotype with the T cell.
  • APC used to form cell hybrids will also preferably, but not necessarily, be derived from the same donor as the T cell. In the production of cell hybrids, either the APC or the IC will be preferably be from, or MHC-compatible with, the donor of the T cell. Alternatively, the APC and/or the IC can share one MHC haplotype (i.e., be semi-allogeneic) with the donor of the T cell.
  • the cells or hybrids used as immunogenic stimuli will frequently be used in the presence of APC of the T cell donor (e.g., in in vivo applications), they can be fully MHC incompatible with the T cell.
  • heat shock proteins bound to antigenic peptide-epitopes derived from antigens e.g., tumor-associated antigens or antigens produced by infectious microorganisms
  • antigens e.g., tumor-associated antigens or antigens produced by infectious microorganisms
  • Heat shock proteins of interest include, without limitation, glycoprotein 96 (gp96), heat shock protein (hsp) 90, hsp70, hsp110, glucose-regulated protein 170 (grp 170) and calreticulin.
  • Immunogenic stimuli can include one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, more) heat shock proteins isolated from tumor cells or infected cells.
  • tumor or infected cells are preferably, but not necessarily, from the same subject (i) whose immune response is to be enhanced by a method of the invention or (ii) from whom T cells (whose response is to be enhanced by a method of the invention) were obtained.
  • the tumor or infected cells can also be obtained, for example, from another individual having the same or a related tumor-type or infection as the subject.
  • the heat shock protein can be isolated from mammalian cells expressing a transcriptosome prepared from tumor cells or inflected cells of interest.
  • Immunogenic stimuli can be derived from a wide range of infectious microorganisms (e.g., bacteria, fungi including yeasts, viruses, and parasites such as protozoan parasites).
  • infectious microorganisms e.g., bacteria, fungi including yeasts, viruses, and parasites such as protozoan parasites.
  • relevant microorganisms include, without limitation, Mycobacteria tuberculosis, Salmonella enteriditis, Listeria monocytogenes, M.
  • microbial proteins include, without limitation, the B subunit of heat labile enterotoxin of E. coli [ Konieczny et al. (2000) FEMS Immunol. Med. Microbiol. 27(4):321-332 ], heat-shock proteins, e.g., the Y. enterocolitica heat shock protein 60 [Konieczny et al. (2000) supra ; Mertz et al. (2000) J. Immunol. 164(3):1529-1537 ] and M. tuberculosis heat-shock proteins hsp60 and hsp 70, the Chlamydia trachomatis outer membrane protein [ Ortiz et al. (2000) Infect. Immun.
  • E. coli outer membrane protein F [ Williams et al. (2000) Infect. Immun. 68(5):2535-2545 ], influenza virus hemagglutinins and neuramindases, retroviral (e.g., HIV) surface glycoproteins (e.g., HIV gp160/120), or retroviral tat or gag proteins.
  • CTL are by virtue of their ability to kill target cells infected with any of a wide variety of intracellular pathogens (e.g., viruses, or intracellular bacteria and protozoans) potent mediators of immunity to such pathogens.
  • intracellular pathogens e.g., viruses, or intracellular bacteria and protozoans
  • helper T cells release a wide variety of cytokines that mediate pathogen-destructive inflammatory reponses.
  • immunogenic stimuli useful in the invention can be any of a wide variety of tumor cells, APC “primed” with tumor cells, hybrid cells (see above), tumor-associated antigens (TAA), peptide-epitopes of such TAA, and APC “primed” with TAA or peptide-epitopes of them.
  • TAA tumor-associated antigens
  • a "TAA” is a molecule (e.g., a protein molecule) that is expressed by a tumor cell and either (a) differs qualitatively from its counterpart expressed in normal cells, or (b) is expressed at a higher level in tumor cells than in normal cells.
  • a TAA can differ (e.g., by one or more amino acid residues where the molecule is a protein) from, or it can be identical to, its counterpart expressed in normal cells. It is preferably not expressed by normal cells. Alternatively, it is expressed at a level at least two-fold higher (e.g., a two-fold, three-fold, five-fold, ten-fold, 20-fold, 40-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, or 15,000-fold higher) in a tumor cell than in the tumor cell's normal counterpart.
  • two-fold higher e.g., a two-fold, three-fold, five-fold, ten-fold, 20-fold, 40-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, or 15,000-fold higher
  • tumors examples include, without limitation, hematological cancers such as leukemias and lymphomas, neurological tumors such as astrocytomas or glioblastomas, melanoma, breast cancer, lung cancer, head and neck cancer, gastrointestinal tumors such as gastric or colon cancer, liver cancer, renal cell cancer, pancreatic cancer, genitourinary tumors such ovarian cancer, vaginal cancer, bladder cancer, testicular cancer, prostate cancer or penile cancer, bone tumors, and vascular tumors.
  • hematological cancers such as leukemias and lymphomas
  • neurological tumors such as astrocytomas or glioblastomas, melanoma
  • breast cancer such as astrocytomas or glioblastomas, melanoma
  • lung cancer head and neck cancer
  • gastrointestinal tumors such as gastric or colon cancer
  • liver cancer liver cancer
  • renal cell cancer pancreatic cancer
  • genitourinary tumors such as ovarian cancer, vaginal cancer, bladder
  • TAA include, without limitation, carcinoembryonic antigen (CEA), prostate specific antigen (PSA), MAGE (melanoma antigen) 1-4, 6 and 12, MUC (mucin) (e.g., MUC-1, MUC-2, etc.), tyrosinase, MART (melanoma antigen), Pmel 17(gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V sequence), Prostate Ca psm, PRAME (melanoma antigen), ⁇ -catenin, MUM-1-B (melanoma ubiquitous mutated gene product), GAGE (melanoma antigen) 1, BAGE (melanoma antigen) 2-10, c-ERB2 (Her2/neu), EBNA (Epstein-Barr Virus nuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6 and E7, p53, lung resistance protein
  • pathogenic agents e.g., tumor cells, infectious microorganisms, or cells infected with infectious agents
  • pathogenic agents e.g., tumor cells, infectious microorganisms, or cells infected with infectious agents
  • an agonistic 4-1BB-binding agent optionally given with one or more non-specific agents such as cytokines (see above)
  • the pathogenic agents do not per se constitute an immunogenic stimulus for the purposes of the invention.
  • an agonistic 4-1BB-binding agent and optionally one or more non-specific agents such as cytokines (see above)
  • pathogenic agents e.g., tumor cells, infectious microorganisms, or cells infected with infectious agents
  • the harbored pathogenic agents do not per se constitute an immunogenic stimulus for the purposes of the invention.
  • the agonistic 4-1BB-binding agent can be an antibody specific for 4-1BB.
  • antibody refers not only to whole antibody molecules, but also to antigen-binding fragments, e.g., Fab, F(ab') 2 , Fv, and single chain Fv fragments. Also included are chimeric antibodies.
  • the antibody can be a polyclonal antibody or a monoclonal antibody (mAb)., e.g., the 2A mAb or the 5.9 and 5.10 mAbs described below.
  • the agonistic 4-1BB-binding agent can be the natural 4-1BB ligand (4-1BBL) or a functional fragment of 4-1BB.
  • a functional fragment of 4-1BB means a fragment of 4-1BB that is shorter than full-length, mature 4-1BBL and has at least 10 % (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% or more) of the ability of full-length mature 4-1 BBL to enhance the response of a T cell to antigen of interest.
  • Methods of testing and comparing the ability of molecules to enhance the response of T cells are known to investigators in the field, e.g., methods (or simple modifications of those) described in the Examples.
  • 4-1BB-binding agents e.g., antibodies
  • bind to a domain of 4-1BB that is identical or overlapping with a domain to which 4-1BBL binds are not agonistic
  • the invention is not limited by any particular mechanism of action. Methods to test for agonistic activity in a candidate 4-1BB-binding agent would be essentially the same as those referred to above for testing the ability of a fragment of 4-1BBL for its ability to enhance the response of T cell to an immunogenic stimulus and thus well-known to those in the art.
  • the agonistic 4-1BB-binding agents can be added to the solution (e.g., blood or culture medium) containing the T cell. Alternatively, it can be secreted by or expressed on the surface of a cell in the vicinity of the T cell, e.g., an APC presenting an alloantigen or a peptide-epitope bound to an MHC molecule on the surface of the APC.
  • a cell in the vicinity of the T cell, e.g., an APC presenting an alloantigen or a peptide-epitope bound to an MHC molecule on the surface of the APC.
  • Such cells can also be tumor cells, infected cells, or the cell hybrids described above.
  • the cell can be, but is not necessarily, the same cell presenting an alloantigen or a peptide-epitope bound to an MHC molecule to the T cell.
  • the methods of the invention require the provision of an exogenous source of the 4-1BB-binding agent. It is understood that where the agonistic 4-1BB-binding agent used in the methods of the invention is one secreted by or expressed on the surface of a cell such as an APC, tumor cell, infected cell, or hybrid cell, it will not be an agonistic 4-1BB-binding agent (e.g., 4-1BBL) naturally expressed by such cells.
  • an agonistic 4-1BB-binding agent e.g., 4-1BBL
  • an agonistic 4-1BB-binding agent where the only source of an agonistic 4-1BB-binding agent is that on the surface of or secreted by an APC, the agonistic 4-1BB-binding agent will be encoded by a recombinant 4-1BB encoding nucleic acid molecule in the APC.
  • fortuitous administration to a subject of an agonistic 4-1BB-binding agent present in, for example, blood, plasma, or serum administered to the subject for, e.g., therapeutic purposes does not per se constitute administration of an agonistic 4-1BB-binding agent for the purposes of the invention.
  • the fortuitous presence of an agonistic 4-1BB-binding agent in culture medium used for immune cell (e.g., T cell) activating cultures does not per se constitute the agonistic 4-1BB-binding agent required to be present in the in vitro methods of activating T cells of the invention.
  • the 4-1BB binding agent can be bound to the floor of a relevant culture vessel, e.g. a well of a plastic microtiter plate.
  • the agonistic 4-1BB-binding agent will preferably, but not necessarily, bind to 4-1BB on the surface of a T cell whose response is enhanced by the methods of the invention.
  • 4-1BB is expressed on cells other than T cells, e.g., natural killer (NK) cells and monocytes [ Melero et al. (1998) Cell. Immunol. 190:167-172 ; Kienzle et al. (2000) Int. Immunol. 12:73-82 ].
  • NK natural killer
  • binding of the T cell or the response of a bystander cell e.g., a B cell cell antibody response
  • binding of a 4-1BB-binding agent to a CD4+ T cell can overcome anergy in a bystander CD4+ T cell or CD8+ T cell that is also exposed to an immunogenic stimulus by, for example, the action of cytokines produced by the CD4+ T cell to which the 4-1BB binding agent binds.
  • binding of a 4-1BB-binding agent to a CD8+ T cell could overcome anergy in a bystander CD8+ T cell or CD4+ T cell exposed to an immunogenic stimulus.
  • Short amino acid sequences can act as signals to direct proteins (e.g., immunogenic stimuli or agonistic 4-1BB-binding agents) to specific intracellular compartments.
  • proteins e.g., immunogenic stimuli or agonistic 4-1BB-binding agents
  • hydrophobic signal peptides e.g., MAISGVPVLGFFIIAVLMSAQESWA (SEQ ID NO:1)
  • KFERQ SEQ ID NO:2
  • other sequences e.g., MDDQRDLISNNEQLP (SEQ ID NO:3) direct polypeptides to endosomes.
  • the peptide sequence KDEL (SEQ ID NO:4) has been shown to act as a retention signal for the ER.
  • Each of these signal peptides, or a combination thereof, can be used to traffic, for example the immunogenic stimuli or agonistic 4-1BB-binding agents to appropriate cellular compartments.
  • Other signal sequences of interest include the HIV tat transduction domain (RKKRRQRR; SEQ ID NO:5), the Antennapedia homeodomain (RQIKIWFPNRRMKWKK; SEQ ID NO:6) and signal sequences derived from fibroblast growth factor [ Lin et al. (1995) J. Biol. Chem. 220:14255-14258 ], transportan [ Pooga et al. (1998) FASEB J.
  • DNAs encoding the polypeptides containing targeting signals can be generated by PCR or other standard genetic engineering or synthetic techniques.
  • the immunogenic T cell stimuli and agonistic 4-1BB-binding agents can have the amino acid sequences of naturally occurring molecules or they can have substitutions. Such substitutions will preferably be conservative substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine, and threonine; lysine, histidine, and arginine; and phenylalanine and tyrosine.
  • Polypeptides useful for the invention also include those described above, but modified for in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the relevant polypeptide in vivo. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake.
  • blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. This can be done either chemically during the synthesis of the peptide or by recombinant DNA technology by methods familiar to artisans of average skill.
  • blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety.
  • the peptide compounds can be covalently or noncovalently coupled to pharmaceutically acceptable "carrier" proteins prior to administration.
  • Peptidomimetic compounds that are designed based upon the amino acid sequences of polypeptides of interest.
  • Peptidomimetic compounds are synthetic compounds having a three-dimensional conformation (i.e., a "peptide motif") that is substantially the same as the three-dimensional conformation of a selected peptide.
  • the peptide motif provides the peptidomimetic compound with the ability to activate an immune response (in the case of immunogenic stimuli) and enhance an immune response (in the case of the agonistic 4-1BB-binding agents).
  • Peptidomimetic compounds can have additional characteristics that enhance their in vivo utility, such as increased cell permeability and prolonged biological half-life.
  • the peptidomimetics typically have a backbone that is partially or completely non-peptide, but with side groups that are identical to the side groups of the amino acid residues that occur in the peptide on which the peptidomimetic is based.
  • Several types of chemical bonds e.g., ester, thioester, thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, are known in the art to be generally useful substitutes for peptide bonds in the construction of protease-resistant peptidomimetics.
  • Molecules useful as immunogenic stimuli and agonistic 4-1BB-binding agents can be produced by any of a wide range of methods known in the art. They can be purified from natural sources (e.g., from any of the pathogenic agents listed herein). Smaller peptides (fewer than 100 amino acids long) and other non-protein molecules can be conveniently synthesized by standard chemical means known to those in the art. In addition, both polypeptides and peptides can be manufactured by standard in vitro recombinant DNA techniques and in vivo transgenesis using nucleotide sequences encoding the appropriate polypeptides or peptides (see Nucleic Acids section below).
  • the transcriptional/translational regulatory elements referred to above include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements that are known to those skilled in the art and that drive or otherwise regulate gene expression.
  • Such regulatory elements include but are not limited to the cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast ⁇ -mating factors.
  • the expression systems that may be used for purposes of the invention include but are not limited to microorganisms such as bacteria (for example, E. coli and B. subtilis ) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing a nucleic acid molecules encoding immunogenic stimuli or agonistic 4-1BB-binding agents; yeast (for example, Saccharomyces and Pichia ) transformed with recombinant yeast expression vectors containing a nucleic acid encoding immunogenic stimuli or agonistic 4-1BB-binding agents; insect cell systems infected with recombinant virus expression vectors (for example, baculovinis) containing a nucleic acid encoding immunogenic stimuli or agonistic 4-1BB-binding agents; plant cell systems infected with recombinant virus expression vectors (for example, cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or transformed with recombinant plasmid
  • Cells transfected or transduced with the expression vectors of the invention can then be used, for example, for large or small scale in vitro manufacture of an immunogenic stimulus or agonistic 4-1BB-binding agent by methods known in the art.
  • methods known in the art In essence, such.methods involve culturing the cells under conditions that maximize production of the polypeptide and isolating the polypeptide from the cells or from the culture medium.
  • the immunogenic stimuli and/or agonistic 4-1BB-binding agents be purified.
  • Methods for purifying biological macromolecules e.g., proteins
  • the degree of purity of the macromolecules can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • a T cell whose response is enhanced, whose anergy is reversed, or in which induction of anergy is prevented by the methods of the invention can be a CD4+ T cell or a CD8+ T cell.
  • the invention is not limited by: (a) the T cell having any particular phenotype (e.g., CD4+ or CD8+) or function (e.g., cytotoxicity, helper activity, immune deviating activity, or suppressive activity); or (b) the MHC molecules by which the T cell is restricted being of any particular class.
  • CD4+ CTL that recognize antigenic peptides bound to MHC class II molecules are known in the art.
  • CD4+ CTL that recognize peptides bound to MHC class I molecules and CD8+ CTL that recognize antigenic peptides bound to MHC class II molecules have also been described.
  • T cells with helper and/or immune deviating activity are CD4+ T cells and recognize antigenic peptides bound to MHC class II molecules, these activities have also been subserved in MHC class I restricted CD8+ T cells.
  • immunosuppressive T cells are CD8+ T cells
  • CD4+ T cells with immunosuppressive activity have also been demonstrated.
  • the methods of the invention are applicable to all these T cells.
  • Preferred responses will be those of MHC class I restricted CTL and MHC class II restricted CD4+ helper/immune deviating T cells. Responses of MHC class I restricted CTL are particularly preferred.
  • the methods of the invention can be performed in vitro .
  • a 4-1BB-binding agent can be added to in vitro assays (e.g., in T cell proliferation assays) designed to test for immunity to an antigen of interest in a subject from which the T cells were obtained. Addition of a 4-1BB-binding agent to such assays would be expected to result in a more potent, and therefore more readily detectable, in vitro response.
  • the methods of the invention will preferably be ex vivo (see below).
  • lymphoid cells consisting of or including T cells obtained from a mammalian subject are cultured with any of the above described immunogenic stimuli and agonistic 4-1BB-binding agents.
  • the lymphoid cells can be from a subject pre-exposed to a relevant antigen (in any of the forms described above); alternatively, the donor of the lymphoid cells need not have been exposed to the antigen.
  • the cultures can also be supplemented with one or move cytokines or growth factors such as, without limitation, interleukin-(IL-)1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-12, IL-13, I-15, interferon- ⁇ (IFN- ⁇ ), tumor necrosis factor- ⁇ (TNF- ⁇ ), granulocyte macrophage colony-stimulating factor (GM-CSF), or granulocyte-colony stimulating factor (G-CSF).
  • the cultures can be "restimulated” as often as necessary.
  • the cultures can also be monitored at various times to ascertain whether the desired level of immune reactivity (e.g., CTL or helper T cell activity) has been attained.
  • lymphoid cells including T cells (CD4+ and/or CD8+ T cells), are isolated from a subject and exposed to one or more immunogenic stimuli and one or more agonistic 4-1BB-binding antibodies in vitro (see above).
  • T cells can be, for example, anergic T cells in which it is desired to reverse anergy.
  • the lymphoid cells can be exposed once or multiply (e.g., 2, 3, 4, 6, 8, or 10 times).
  • the level of immune activity (e.g., CTL activity) in the lymphoid cells can be tested after one or more exposures. Once the desired activity and level of that activity is attained, the cells are reintroduced into the subject (or another subject) via any of the routes listed herein.
  • the therapeutic or prophylactic efficacy of this ex vivo approach is dependent on the ability of the ex vivo activated lymphocytes to exert, directly or indirectly, a neutralizing or cytotoxic effect on, for example, infectious microorganisms, host cells infected with microorganisms, or tumor cells.
  • An alternative ex vivo strategy can involve transfecting or transducing cells obtained from a subject with one or more polynucleotides encoding one or more T cell specific immunogenic stimuli and one or more agonistic 4-1BB-binding antibodies. The transfected or transduced cells are then returned to the subject or another subject.
  • hemopoietic cells e.g., bone marrow cells, macrophages, monocytes, dendritic cells, or B cells
  • they could also be any of a wide range of types including, without limitation, fibroblasts, epithelial cells, endothelial cells, keratinocytes, or muscle cells in which they act as a source of the said one or more immunogenic stimuli and one or more agonistic 4-1BB-binding agents for as long as they survive in the subject.
  • hemopoietic cells that include the above APC, would be particularly advantageous in that such cells would be expected to home to, among others, lymphoid tissue (e.g., lymph nodes or spleen) and thus the immunogenic stimuli and agonistic 4-1BB-binding agents would be produced in high concentration at the site where they exert their effect, i.e., enhancement of an immune response.
  • lymphoid tissue e.g., lymph nodes or spleen
  • the APC expressing the exogenous can be, but are not necessarily, the same APC that presents an alloantigen or antigenic peptide to the relevant T cell.
  • the agonistic 4-1BB-binding agents can be secreted by the APC or expressed on its surface. Prior to administering recombinant APC to a subject, they can optionally be exposed to the above-listed sources of antigens or antigenic peptides of interest, e.g., those of tumors or infectious microorganisms. The same genetic constructs and trafficking sequences described for the in vivo approach can be used for this ex vivo strategy. Furthermore, tumor cells or hybrid cells produced by fusion of APC (e.g., dendritic cells) and tumor cells can be transfected or transformed by one or more vectors encoding said one or more agonistic 4-1BB-binding agents.
  • APC e.g., dendritic cells
  • Such cells are then administered to a subject with the relevant cancer where, due to their expression of the exogenous agonistic 4-1BB-binding agents (on their cell surface or by secretion), they can stimulate enhanced tumoricida T cell immune responses.
  • an agent e.g., ionizing irradiation or mitomycin C
  • tumor cells which, after transfection or transformation with said agonistic 4-1BB-binding agent encoding nucleic acids, are administered to a subject with cancer can have been obtained from an individual other than the subject.
  • tumor cells used for the production of hybrid cells that express said recombinant agonistic 4-1BB-binding agents and are administered to a subject with cancer can have been obtained from an individual other than the subject.
  • the ex vivo methods can include the steps of harvesting cells (e.g., tumor cells or APC) from a subject, culturing the cells, transducing them with one or more expression vectors, and maintaining the cells under conditions suitable for express ion of the said immunogenic stimuli and/or said agonistic 4-1BB-binding agents. These methods are known in the art of molecular biology.
  • the transduction step is accomplished by any standard means used for ex vivo gene therapy, including calcium phosphate, lipofection, electroporation, viral infection, and biolistic gene transfer. Alternatively, liposomes or polymeric microparticles can be used.
  • Cells that have been successfully transduced are then selected, for example, for expression of the coding sequence or of a drug resistance gene.
  • the cells may then be lethally irradiated (if desired) and injected or implanted into the patient.
  • the methods can include the additional step of making the above-described cells hybrids that are injected or implanted into the patient
  • An immunogenic stimulus can be provided in the form of a peptide-epitope and agonistic 4-1BB-binding antibody in the form of either a nucleic acid encoding it or cells transformed with a nucleic acid encoding it.
  • the invention features antibodies that bind specifically to human and mouse 4-1BB.
  • Such antibodies can be polyclonal antibodies present in the serum or plasma of animals (e.g., mice, rabbits, rats, guinea pigs, sheep, horses, goats, cows, or pigs that have been immunized with the relevant 4-1BB polypeptide (or a peptide fragment thereof) using methods, and optionally adjuvants, known in the art.
  • Such polyclonal antibodies can be isolated from, for example, serum, plasma, or ascites by methods known in the art.
  • Monoclonal antibodies (mAb) that bind to 4-1BB polypeptides or fragments are also encompassed by the invention. These mAbs include those produced by the 2A, 5.9, and 5.10 hybridomas (see Examples).
  • the resulting antibody can be produced by a number of in vivo and in vitro methods known in the art.
  • the hybridoma can be cultured in vitro in a suitable medium for a suitable length of time, followed by the recovery of the desired antibody from the supernatant.
  • the length of time and medium are known or can be readily determined.
  • recombinant antibodies specific for 4-1BB such as chimeric and humanized monoclonal antibodies comprising both human and non-human portions
  • chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, using methods described in Robinson et al., International Patent Publication PCT/US86/02269 ; Akira et al., European Patent Application 184, 187 ; Taniguchi, European Patent Application 171,496 ; Morrison et al., European Patent Application 173,494 ; Neuberger et al., PCT Application WO 86/01533 ; Cabilly et al., U.S. Patent No.
  • antibody fragments and derivatives which contain at least the functional portion of the antigen binding domain of an antibody that binds specifically to 4-1BB.
  • Antibody fragments that contain the binding domain of the molecule can be generated by known techniques.
  • fragments include, but are not limited to: F(ab') 2 fragments that can be produced by pepsin digestion of antibody molecules; Fab fragments that can be generated by reducing the disulfide bridges of F(ab') 2 fragments; and Fab fragments that can be generated by treating antibody molecules with papain and a reducing agent. See, e.g., National Institutes of Health, 1 Current Protocols In Immunology, Coligan et al., ed.
  • Antibody fragments also include Fv (e.g., single chain Fv (scFv)) fragments, i . e ., antibody products in which there are few or no constant region amino acid residues.
  • Fv e.g., single chain Fv (scFv)
  • An scFv fragment is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the ScFv is derived. Such fragments can be produced, for example, as described in U.S. Patent No. 4,642,334 .
  • Tumor models and peptides C3 cells generated from HPV-16-/EJras-transformed C57BL/6 (B6) mouse embryo cells [ Feltkamp et al. (1993) Eur. J. Immunol. 23:2242-2249 ], were a gift from Dr. W. Martin Kast (Loyola University, Chicago, IL).
  • a line of EL4 cells (EL4E7) transfected with cDNA encoding the human papilloma virus-16 (HPV-16) E7 polypeptide [ Tindle et al. (1995) Clin. Exp. Immunol. 101:265-271 ] was a gift from Dr. Germain J.P. Fernando (University of Queensland, Brisbane, Australia).
  • the TC-1 cell line [ Liu et al. (1996) Cancer Res. 56:21-6 ] was a gift from Dr. T.C. Wu (Johns Hopkins University, Baltimore, MD) and the B16-F10 melanoma line [ Dranoff et al. (1993) Proc. Natl. Acad. Sci. USA 90:3539-3543 ] was a gift from Dr. Glenn Dranoff (Dana-Farber Cancer Institute, Boston, MA).
  • the EL4, RMA-S and S49.1 murine T cell lymphoma lives were of B6 origin and were purchased from the American Type Culture Collection (Manassas, VA).
  • P815R The regressor P815 mastocytoma (P815R), which has been previously described [ Nieland et al. (1999) J. Cell Biochem 73:145-152 ], was obtained from Dr. W. Martin Kast, Loyola University, Chicago, IL. All cell lines were maintained in a complete tissue culture medium of RPMI 1640 (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 25 mM HEPES, 2mM glutamine, 100 U/ml penicillin G and 100 ⁇ g/ml streptomycin sulfate.
  • FBS fetal bovine serum
  • the E7 peptide (RAHYNIVTF) (SEQ ID NO:7) contained the minimal H-2D b -restricted CTL epitope [ Feltkamp et al. (1993) Eur. J. Immunol. 23:2242-2249 ] of HPV-16 E7 protein.
  • the trp-2 peptide (SVYDFFVWL) (SEQ ID NO:8) is a H-2K b -restricted epitope first identified in the B16 melanoma [ Bloom et al. (1997) J. Exp. Med.185:453-459 ; Schreurs et al. (2000) Cancer Res. 60:6995-7001 ].
  • the Vp2 control peptide (FHAGSLLVFM) (SEQ ID NO:9) contains an H-2D b -restricted CTL epitope derived from the Theiler's Murine Encephalomyelitis Virus [ Johnson et al. (1999).J. Virol. 73:3702-3708 ].
  • the OVA (257-264) peptide (SIINFEKL) (SEQ ID NO:10) (referred to in the OT-1 T cell anergy experiments described in Examples 10-13 as the "OVA peptide") is a H-2K b -restricted CTL epitope derived from chicken ovalbumin [ Curtsinger et al. (1998) J. Immunol.
  • the OVA (55-62) peptide (KVVRFDKL) (SEQ ID NO: 11) used as a "control peptide" in the OT-1 T cell anergy experiments described in Examples 10-13 is a H-2K b -restricted CTL epitope from chicken ovalbumin [E1- Shami et al. (1999) Eur. J. Immunol. 29:3295-3301 ]
  • the P1A (35-43) peptide (LPYLGWLVF) (SEQ ID NO:12) is an H-2L d restricted CTL epitope.
  • peptides were synthesized by the Mayo Molecular Biology Core Facility and the purity of the peptides was >90% by reverse-phase HPLC purification.
  • the peptides were dissolved in dimethyl sulfoxide (DMSO) and reconstituted in phosphate buffered saline (PBS) to a final concentration of 1 mg/ml (5% DMSO) for administration to mice.
  • DMSO dimethyl sulfoxide
  • PBS phosphate buffered saline
  • mice Female B6 mice were purchased from the National Cancer Institute (Frederick, MD). Age-matched mice, 6-10 weeks old, were used for all experiments. Tumor cells in 0.1 ml of PBS were injected subcutaneously (s.c.) into the right shaved flanks. Mice were given 1 x 10 6 C3 cells or 4 x 10 6 EL4E7 cells. Tumor size (the average of two perpendicular diameters in mm) was measured weekly as previously described [ Tamada et al. (2000) Nature Med. 6:283-289 ].
  • mice For lung metastases models, 1 x 10 4 TC-1 or 1 x 10 5 B16-F10 cells were injected in 0.5 ml of Hank's Buffered Salt Solution into the tail vein of mice. Mice bearing subcutaneous tumors were immunized intradermally (i.d.) at a site contralateral to the tumor with 50 ⁇ g of peptide emulsified in incomplete Freund's adjuvant (IFA) (Sigma Chemicals, St. Louis, MO). Mice bearing lung metastases were immunized bilaterally i.d. with a total of 100 ⁇ g of peptide emulsified in IFA. Antibodies administered to mice were injected intraperitoneally (i.p.) in 0.5 ml of PBS.
  • IFA incomplete Freund's adjuvant
  • OT-1 transgenic mice which have been previously described [ Strome et al. (2002) Cancer Research 62:1884-1889 ], were obtained from Dr. Larry Pease, Mayo Clinic, Rochester, MN. All the CD8+ T cells of the OT-1 mice express an antigen specific T cell receptor (TCR) specific for the OVA (257-264) peptide bound to the murine H-2K b MHC class I molecule.
  • TCR antigen specific T cell receptor
  • cDNA encoding the extracellular domain of mouse 4-1BB was amplified from cDNA produced from RNA isolated from concanavilin A-activated spleen cells using sequence specific primers and was fused to the CH 2 -CH 3 domain of mouse IgG2a in the expression plasmid pmIgV [ Chapoval et al. (2000) Nature Med. 6:283-289 ].
  • the resulting expression vector was transfected into CHO cells.
  • the protein in the culture supernatants of a transfected clone was purified using a HiTrap Protein G-Sepharose column (Amersham Pharmacia Biotech, Piscataway, NJ) and dialyzed into lipopolysaccharide-free PBS.
  • a rat monoclonal antibody (mAb) against 4-1 BB was generated by immunizing a Lewis rat (Harlan Sprague Dawley, Indianapolis, IN) with mouse 4-1BBIg.
  • Hybridomas were produced by fusing rat spleen cells with mouse Sp2/0 myeloma cells and the culture supernatants were screened by ELISA.
  • the hybridoma secreting the mAb 2A was selected for further experiments.
  • Hybridoma 2A was grown in RPMI 1640 supplemented with 10% low IgG FBS (Life Technologies) and 25 mM HEPES and supernatant was harvested and concentrated using a tangential flow miniplate concentrator (Millipore, Bedford, MA).
  • the 2A mAb was purified from the concentrated supernatant using a 5 ml HiTrap Protein G-Sepharose column (Amersham Pharmacia Biotech, Piscataway, NJ). The purified mAb was dialyzed against PBS and concentrated using a Centriprep concentrator (Millipore, Bedford. MA). The isotype of the 2A mAb was determined using biotinylated, isotype-specific antibodies (Caltag Laboratories, Burlingame, CA). It was found to be an IgG2a antibody with kappa light chains.
  • mAb specific for mouse CD3, CD28, 4-1BB, fluorescein isothiocyanate-(FITC-) conjugated CD8, CD69, CD25, CD49A, and isotype control mAb, and Cy-Chrome-conjugated CD8 mAb were purchased from PharMingen (San Diego, CA).
  • the FITC-conjugated goat anti-rat IgG antibody was purchased from Biosource International (Camarillo, CA).
  • Rat IgG antibodies (Sigma Chemical, Gilbertsville, PA) were used as controls.
  • H-2D b major histocompatibility complex (MHC) class I molecule bound to either the E7 peptide (H-2D b -E7) or the control Vp2 peptide (H-2D b -Vp2) were prepared as previously described [ Johnson et al. (1999) J. Virol. 73:3702-3708 ]. Briefly, H-2D b ⁇ -chain and human ⁇ 2 -microglobulin were isolated from a bacterial expression system and subsequently folded in the presence of excess peptide. The folded monomeric complexes were desalted and biotinylated.
  • the monomeric complexes were conjugated to strepavidin labeled with the fluorescent dye, phycoerythin (PE), thereby forming fluorescent tetrameric complexes.
  • PE phycoerythin
  • the PE-labeled tetramers generated were then purified by size exclusion gel filtration.
  • a tetramer composed of four mouse H-2K b molecules bound to the OVA (257-264) peptide and labeled with PE (sometimes referred to below as the "OVA tetramer") was obtained from the NIH tetramer core facility (Atlanta. GA).
  • T cell costimulation assay The method used to assay costimulatory activity of mAb was described previously [ Tamada et al. (2000) Nature Med. 6:283-289 ].
  • nylon wool (NW)-purified mouse splenic T cells (2.5 x 10 6 /ml) were added to 96-well plates which had been coated with a mAb against CD3 (0.1 ⁇ g/ml) and the indicated concentrations of rat IgG or mAb 2A.
  • the proliferation of T cells was assessed by the addition of 1 ⁇ Ci/well of [ 3 H]-thymidine ( 3 H-TdR) to the 3-day cultures 15 hours before harvesting of the cultures onto fiber glasstilters.
  • 3 H-TdR incorporated into the T cells was measured in a MicroBeta TriLux liquid scintillation counter (Wallac, Turku, Finland).
  • T cells were positively selected using FITC conjugated mAbs against CD4 and CD8, metal microbeads coated with antibody specific for FITC, and a magnet as instructed by the manufacturer (Miltenyi Biotec, Auburn, CA). The purity of the isolated T cells was routinely greater than 95%, as assessed by flow cytometry using a mAb against CD3. Purified T cells (2.5 x 10 6 cells/ml) from mouse spleens were stimulated in the wells of 24-well tissue culture plates coated with mAbs against CD3 (5 ⁇ g/ml) and CD28 (1 ⁇ g/ml).
  • T cells were collected and stained for 30 min at 4°C with 1 ⁇ g mAb 2A, either alone or in the presence of 4-1BBIg (2 ⁇ g/ml), in 50 ⁇ l PBS supplemented with 3% FBS and 0.02% azide. The cells were washed and incubated an additional 30 min at 4°C with FITC-conjugated goat antibody specific for rat IgG. After washing the cells were fixed in 1% paraformaldehyde and fluorescence was analyzed with a FACS (Becton Dickinson, Mountain View, CA). S49.1 cells were stained in a similar fashion.
  • FACS Becton Dickinson, Mountain View, CA
  • Tumor-draining lymph nodes from immunized mice were harvested on day 7 and stained with PE-labeled H-2D b- E7 or H-2D b -Vp2 tetrameric complexes and FITC-conjugated CD8 as previously described [ Johnson et al. (1999) J. Virol. 73:3702-3708 ].
  • Five x 10 6 TDLN cells were incubated with 2.5 x 10 -5 UV-irradiated C3 cells for 4 days. Cells were subsequently stained with the PE-labeled tetramers and FITC-conjugated CD8. After extensive washing, cells were re-suspended in PBS with 750 ng/ml propidium iodide. Gates were drawn to include viable CD8 + cells only.
  • T cells were purified from spleens and lymph nodes of mice using microbead coated with antibody specific for Thy1.2 according to the manufacturers instructions (Miltenyi Biotec, San Diego, CA). Purified Thy1.2+ cells were subsequently stained with the OVA tetramer described above. Positively stained cells were sorted using a FACSVantage Flow Cytometry System (B D Immunocytometry Systems, San Jose, CA). At least 90% of the sorted cells were both OVA tetramer positive and CD8+.
  • effector cells were obtained by co-culturing draining LN cells with irradiated C3 cells for 4 days. Effector cells were harvested from the cultures and tested for CTL activity using a standard 4-hr 51 Cr release assay with tumor cell targets at the indicated effector to target cell (E:T) ratios. Peptide-pulsed target cells were generated by culturing the target cells with 10 ⁇ g/ml of the peptide at 28°C for 18 hours prior to use. The CTL assay used for the OT-1 T cell anergy experiments was performed similarly using the target cells indicated below.
  • Murine DC were prepared from bone marrow. Mice were sacrificed and dipped in 70% ethanol (EtOH). After removing excess EtOH, the hind limbs were exposed and the hip joint dislocated. Muscle parenchyma was removed and the bones placed briefly in 70% EtOH and then in complete medium (CM; RPMI 1640, 10% heat inactivated FBS (Hyclone Laboratories, Inc., Logan, UT), Fungizone (0.5 ⁇ g/ml), ⁇ -ME (2 x 10 -5 M), sodium pyruvate (1 mM), non-essential amino acids (0.1 mM), penicillin and streptomycin (100 ⁇ g/ml), glutamine (2 mM) and Gentamycin (50 ⁇ g/ml)).
  • CM complete medium
  • Both ends of the bones were cut to expose the marrow and a 3cc syringe (filled with CM) with a 25 gauge needle was used to eject the marrow into a 10mm cell culture dish, containing CM.
  • the cells were separated from stromal components by straining through a steel sieve and, after pelleting by centrifugation, were resuspended in 1.0 ml medium of containing 10 ⁇ g/ml of anti-class II (I-A b ) mAb, anti-Mac 3 mAb, anti-CD8a mAb (HO2.2), anti-CD45R (B220) mAb, anti-CD3e mAb, and anti-GR-1 mAb (all from PharMingen, Inc.
  • mice Induction of T cell anergy 3-7 x 10 6 lymph node and spleen cells from OT-1 mice were injected intravenously (i.v.; tail vein) into wild type B6 mice in 0.5 ml Hanks balanced salt solution (HBSS) (Cellgro, Herndon, Virginia). 12-24 hours later, experimental mice were given 0.5 mg of OVA (257-264) peptide i.v. in 0.5 ml total volume, while control mice were given OVA (55-62) peptide (or PBS alone in some experiments) in a similar fashion. On the day of peptide administration, and again 3 days later, mice were given 100 ⁇ g of either rat IgG or anti-CD 137 mAb intraperitoneally (i.p.).
  • HBSS Hanks balanced salt solution
  • mice were sacrificed at various time points following peptide administration and the total number of OT-1 cells present in the spleen and lymph nodes of each mouse was determined by OVA tetramer staining.
  • spleens were harvested from the mice. After lysing red blood cells in Ack lysis buffer, the spleen cells were resuspended in RPMI tissue culture medium supplemented as described above for the medium used to culture tumo cell lines and plated in triplicate into the wells of a 96-well plate at a density of 5.5x10 5 cells per well in final volume of 200 ⁇ l per well.
  • One group of cells was unstimulated while a second group was restimulated with 1 ng/ml of OVA peptide.
  • the frequency of OVA-specific T cells was determined using the H-2K b -OVA (257-264) tetramer.
  • the absolute number of OT-1 cells added to each well could be calculated prior to restimulation in vitro .
  • Supernatants were collected from the wells in each group 48 and 72 hours after restimulation and IL-2 (48 hr) and IFN- ⁇ (72 hr) production was measured by sandwich ELISA following the manufacturer's instructions (PharMingen, San Diego, California).
  • T cells The proliferation of T cells was assessed by the addition of 1 ⁇ Ci/well [ 3 H]-thymidine during the last 15 hours of the 3-day culture. [ 3 H]-thymidine incorporation was measured in a MicroBeta TriLux liquid scintillation counter (Wallac, Turku, Finland). Antigen specific proliferation or cytokine production per OT-1 cell was calculated by subtracting any nonspecific proliferation (or cytokine production) observed in the unstimulated groups from the proliferation (or cytokine production) observed in the peptide stimulated groups. This was then divided by the number of OT-1 cells (10 3 ) initially present in the well prior to restimulation to derive the net change in cpm ( ⁇ cpm) per 10 3 OT-1 cells.
  • OT-1 cells were adoptively transferred into wild type recipients. Anergy was induced by the intravenous administration of 0.5 mg OVA peptide as described above. Alternatively, mice received only control peptide or PBS and were considered "na ⁇ ve" at the time of rechallenge with the antigen. Ten days later mice were given 0.5 mg OVA peptide or control peptide intravenously. Mice received 100 ⁇ g of either rat IgG or anti-CD137. Mice were sacrificed at various time points following rechallenge with the OVA peptide and the number of OT-1 cells present in the spleen and lymph nodes of each mouse was determined by tetramer analysis, as before.
  • mAb 2A binds specifically to the mouse T cell lymphoma S49.1 that constitutively expresses 4-1BB (as demonstrated by staining with 1AH2, a commercially available anti-4-1BB mAb ( Fig. 1B )).
  • Immobilized mAb 2A also enhanced T cell proliferation in a dose-dependent fashion in the presence of a suboptimal dose of anti-CD3 mAb ( Fig. 1C ). Therefore, 2A is a costimulatory mAb similar to others previously described [ Melero et al. (1997) Nature Med. 3:682-685 ; Shuford et al. (1997) 186:47-55].
  • EL4E7 is a thymoma transfected to express the HPV-16 E7 gene and C3 is an embryonic epithelial cell line transformed with HPV-16 and the ras oncogene. Since both tumor lines express the E7 gene of HPV-16, CTL responses to the E7 gene product can be monitored.
  • groups of mice bearing established EL4E7 or C3 tumors were injected i.p. with 2A (100 ⁇ g) at days 7 and 10. As shown in Fig.
  • mice bearing C3 tumors were first inoculated subcutaneously (s.c.) with C3 cells and subsequently (3-7 days later) treated with mAb 2A or control rat IgG. Seven days later, TDLN were harvested, re-stimulated in vitro with irradiated C3 cells, and the CTL activity of cells harvested from cultures was tested in a standard 51 Cr release assay. As shown in Fig.
  • the frequency of E7 (49-57) specific T cells in C3 TDLN was determined by double staining with FITC conjugated anti-CD8 mAb and PE-labeled E7 tetramer. Consistent with the findings on CTL activity, less than 0.1 % of CD8 + T cells in TDLN from C3-bearing mice were E7-specific, even after in vitro re-stimulation with irradiated C3 cells. This value represents a threshold of "undetected CTL" in the assay since similar results were also obtained using cells from na ⁇ ve mice. Furthermore, treatment with the 2A mAb failed to expand E7-specific CTL in C3 TDLN ( Fig. 4A, B ).
  • E7-specific CTL activity was examined in the mice after immunization with the E7 (49-57) peptide that contains a H-2D b restricted CTL epitope. Seven days after peptide immunization, draining LN were harvested, restimulated with irradiated C3 cells and the frequency of E7 specific CD8 + T cells was determined using the fluorescent E7 tetramer. Immunization with the E7 peptide caused a significant increase of cells that bound the E7 tetramer. Such cells were not detectable after immunization with a control Vp2 peptide.
  • E7-specific CTL Treatment with mAb 2A resulted in a further increase in the frequency of E7-specific CTL ( Fig. 5A ). Similar results were obtained by immunization of naive mice ( Fig. 5B ). Therefore, E7-specific CTL are present in C3-bearing mice, but are neither activated nor deleted by the C3 cells. Thus E7-specific CTL ignore antigens presented by the C3 tumor. In addition, anti-4-1BB mAb alone is unable to break this unaware state.
  • mice On the day of immunization and 3 days later, mice were given 100 ⁇ g of mAb 2A or a control rat IgG i.p. Tumor size was assessed weekly. Data shown was pooled from several experiments. b 21 days following treatment, the mean tumor diameter was calculated for those tumors which had failed to completely regress. c The Unpaired Student's T-test was used to calculate p-values comparing the mean tumor diameter of the treatment group which received both the E7 peptide and mAb 2A with those of the control groups.
  • the TC-1 tumor line is derived from primary lung epithelial cells co-transformed with both the HPV-16 E6, HPV-16 E7 and ras oncogenes [ Liu et al. (1996) Cancer Res. 56:21-6 ]. Therefore, the E7 peptide could be used as an immunogen.
  • B16-F10 is a highly metastatic melanoma line, which presents the H-2K b -restricted trp-2 peptide [ Dranoff et al. (1993) Proc. Natl. Acad. Sci. USA 90:3539-3543 ; Bloom et al. (1997) J. Exp.
  • mice were injected intravenously (i.v.) with 10 4 TC-1 cells to establish lung metastases.
  • the mice were injected s.c. with the E7 peptide and i.p. with the 2A mAb (COPP).
  • COPP 2A mAb
  • the administration of mAb 2A alone was insufficient to prolong survival in the tumor-bearing mice, as all of the mice died within 20 days.
  • E7 peptide immunization alone did prolong survival, although all of the mice were dead by day 35.
  • COPP treatment trp-2 peptide and mAb 2A led to a significant survival advantage for all the mice and long-term survival (>90 days) in 20% of treated mice. Therefore, combined treatment with an antigenic, MHC class I restricted peptide and anti-4-1BB mAb (COPP) may be therapeutic for established, poorly immunogenic tumors.
  • the 2A mAb (or control rat IgG) was administered three days after each test vaccination. Tumor growth was measured in a blinded fashion to determine therapeutic efficacy.
  • Fig. 8 80% of animals treated with rat IgG alone developed tumors (group 3; Fig. 8 top right panel).
  • animals treated with tumor "primed" C and rat IgG (group 1; Fig. 8 , bottom right panel) or mAb 2A alone (group 4; Fig 8 , top left panel) there was therapeutic efficacy in 3 out of 5 mice.
  • a strategy similar to that described above for generation of mAbs specific for murine 4-1BB was used.
  • a soluble 4-1BB fusion protein composed of an extracellular fragment of human 4-1BB and the CH3-CH3 domain of human IgG1 was engineered. Mice immunized with this fusion protein produced polyclonal antibodies to 4-1BB. Spleens from these animals were fused with SP2/0 mouse myelomas to produce hybridomas that were screened by fluorescence flow cytometry with 293 cells transfected with cDNA encoding human 4-1BB. Two hybridomas (5.9 and 5.10) producing TgG1 mAbs showed human 4-1BB-specific staining.
  • Example 7 In order to determine whether the two anti-human 4-1BB mAbs described in Example 7 have the ability costimulate a T cell response, a standard in vitro costimulation assay was performed. Briefly, purified human T-cells were cultured with (a) antibody specific for human CD3 coated onto the well-bottoms of 96-well tissue culture plates at various concentrations and (b) varying concentrations of the 5.9 mAb, the 5.10 mAb, or normal mouse IgG, all also coated (at a concentration of 10 ⁇ g/ml) onto the well-bottoms of the 96-well tissue culture plates. 3 H-TdR incorporation measured after a 48-hour culture period was used to determine T cell proliferation. ( Fig. 9 ). Both anti-human 4-1BB stimulated significant T cell proliferation, thereby demonstrating the T cell costimulatory potential of these antibodies.
  • mice were injected i.v. with 5 x 10 5 B16-F10 melanoma cells in order to generate disseminated metastases. On days 3, 7, and 11 the mice were immunized s.c.
  • B16-GM-CSF murine GM-CSF
  • mice injected i.p. with 100 ⁇ g of either rat IgG or mAb 2A as indicated below.
  • the B16-GM-CSF cells were generated essentially as described in Dranoff et al. (1993) Proc. Natl. Acad. Sci. USA 90:3539-3543 .
  • mice were monitored for survival ( Fig. 10 ). Mice immunized with B-16-GM-CSF and mAb 2A showed enhanced survival over mice immunized with B 16-GM-CSF and rat IgG.
  • Intravenous (i.v.) delivery of antigen is an established approach to induce anergy in CD4 + T cells [ Vaiserskikh et al. (2001) Transplantation 72:685-693 ; Thorstenson et al. (2001) J. Immunol. 167:188-195 ; and Bercovici et al. (1999) Eur. J. Immunol. 29:345-354 ].
  • antigen-specific T cells exposed to a high dose of soluble antigen become unresponsive to the antigen; this is demonstrated by the inability of the T cells to proliferate and secrete IL-2 upon exposure to the antigen in vitro in the presence of APC [ Jacobs et. al. (1994) Immunology 82:294-300 ].
  • This approach was adapted by the inventors to examine the induction of anergy in CD8 + T cells.
  • CD8 + T cells were OVA tetramer-positive (i.e., were OT-1 T cells) in the mice treated with control peptide
  • the proportion of CD8 + T cells that were OT-1 T cells in the OVA-treated mice was 6.8% ( Fig. 11 ).
  • OT-1 cell blastogenesis was also observed in the OVA-treated mice, as demonstrated by the increase in forward scatter of the cells from these mice by FACS analysis.
  • more than 50% of the OT-1 cells in the OVA-treated mice expressed the T-cell activation markers, CD69 and CD25 ( Fig. 11 ).
  • mice treated as described above were rechallenged i.v.10 days later with either a control peptide or the OVA peptide.
  • naive mice i.e., mice that had not received the initial injection of OVA peptide
  • spleens and lymph nodes were harvested from the mice and the total numbers of OT-1 cells in pooled spleen and lymph node samples were determined by OVA tetramer staining. A greater than 4-fold expansion was observed in the na ⁇ ve mice treated with OVA.
  • OT-1 cells retained their effector function following the induction of anergy
  • splenocytes from B6 mice injected with OT1 T cells and a single dose of OVA as described above were restimulated with OVA peptide in vitro.
  • the OT-1 cells pre-exposed to OVA were incapable of proliferating in vivo , they secreted IFN- ⁇ upon restimulation with OVA in vitro at a level similar to naive OT-1 T cells ( Fig. 12C ).
  • Example 11 CD137 signaling prevents the induction of OT-1 T cell anergy
  • mice Following adoptive transfer of OT-1 cells into naive B6 mice and the administration of OVA peptide, mice were injected i.p. with either a CD137 mAb (clone 2A) or control rat IgG. Mice were sacrificed at various time points up to 21 days following injection of the OVA peptide and the total number of OT-1 cells present in the spleens and lymph nodes in each group of mice was determined by OVA tetramer staining. As shown in Fig. 13A , treatment with anti-CD137 mAb following OVA administration led to an approximately ten-fold increase in the number OT-1 cells compared to those mice that received the control rat IgG.
  • mice to which OT-1 T cells had been adoptively transferred and that were injected with either PBS, OVA peptide and anti-CD137 mAb, or OVA peptide and control rat IgG were sacrificed ten days following peptide (or PBS) administration.
  • the number of OT-1 cells present in the pooled splenocytes from each group of mice was determined by OVA tetramer staining.
  • the splenocytes were subsequently restimulated in vitro with an optimal concentration of OVA peptide.
  • OT-1 proliferation and IL-2 secretion were measured 72 and 48 hours later, respectively.
  • OT-1 T cells The proliferation of OT-1 T cells, as measured by [ 3 H]-thymidine incorporation, was determined on a per cell basis. Unlike the naive OT1 T cells from mice that had received PBS, OT-1 cells from mice that were given the OVA peptide and control rat IgG failed to proliferate following in vitro restimulation ( Fig. 13B ). Furthermore, FACS analysis demonstrated that virtually 100% of the OT-1 T cells from the mice previously exposed to OVA peptide expressed the late activation marker VLA-4, in contrast to the OT-1 cells in the control peptide-treated mice ( Fig. 13C ). In sharp contrast, proliferation was observed in OT-1 T cells from those mice that received anti-CD137 mAb and OVA peptide.
  • IL-2 production was not observed following restimulation of the anergic OT-1 cells from OVA peptide and anti-CD 137 mAb treated mice, a finding consistent with their lack of proliferation ( Fig. 13B ) whereas OT-1 cells from the anti-CD137 treated mice secreted IL-2 to an extent comparable with naive OT-1 ( Fig. 3B ); this finding provided further evidence that CD 137 signaling prevented the induction of T cell anergy.
  • mice were injected i.v. with OVA peptide as described above in order to induce anergy in the OT-1 T cells, Ten days later, the mice were injected i.p. with anti-CD137 mAb together with OVA peptide. Mice were sacrificed 2, 5 and 10 days following treatment and the total number of OT-1 T cells was determined by OVA tetramer staining. As shown in Fig.
  • OVA peptide OT-1 T cell anergic mice were prepared as described above. Ten days after the injection of OVA peptide, the mice were given various combinations of control peptide or OVA peptide and control rat IgG or anti-CD137 mAb. Three days later, the mice were sacrificed and the total number of OT-1 cells determined. OT-1 cell expansion was only observed in the group of mice that received both OVA peptide and anti-CD137 mAb ( Fig. 14B ).
  • OT-1 T cells were sorted by FACS following staining with the OVA tetramer from the OVA-tolerized mice 5 days after challenge with OVA peptide and CD 137 mAb and were used as effector cells in a standard 4-hour 51 Cr-release assay for cytotoxicity against OVA-pulsed EL4 and control EL4 cells ( Fig. 14D ; "Anergic (OVA + 2A)").
  • OT-1 cells were identically sorted from mice that, instead of initially being injected with the OVA peptide, were injected with the control peptide and then 10 days later were challenged with the OVA peptide and either rat IgG ("OVA + rat IgG") or anti-CD137 mAb ("OVA + 2A"); the cell sorting cytotoxicity testing, as for the experimental group, were performed 5 days after the peptide challenge and mAb/control IgG treatment. As shown in Fig. 14D , approximately 20% specific lysis was observed at an E:T ratio of 10 to 1 in those mice that had received the control IgG.
  • P815R is a clonal variant of highly tumorigenic P815 mastocytoma, which unlike the parent P815 tumor, regresses spontaneously after inoculation into syngeneic DBA/2 mice [ Nieland et al. (1999) J. Cell Biochem. 73:145-152 ].
  • Administration of a peptide epitope of P1A, a non-mutated self tumor antigen [ Van den Eynde et al. (1991) J. Exp. Med.
  • mice were first tolerized with P1A peptide in IFA for 10 days and were subsequently challenged with P815R cells. Three days following tumor challenge, mice were given control rat IgG or anti-CD137 mAb. In mice treated with the control rat IgG, 90% of the mice developed progressively growing tumors ( Fig. 15B , left panel). In contrast, anti-CD137 mAb treatment led to tumor regression in 100% of mice ( Fig. 15B , right panel).

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Claims (70)

  1. (a) Antigène pour lequel un récepteur de cellule T est spécifique, et
    (b) anticorps agoniste liant 4-1BB,
    destinés à une utilisation sous la forme d'un médicament ou d'une combinaison de médicaments afin de générer une réponse immunitaire améliorée chez un sujet.
  2. Antigène et anticorps selon la revendication 1, où l'antigène est (a) un antigène associé à une tumeur (TAA) ou (b) un fragment fonctionnel d'un TAA.
  3. Antigène et anticorps selon la revendication 1, où l'antigène est un polypeptide.
  4. Antigène et anticorps selon la revendication 2, où le TAA est une molécule produite par une cellule cancéreuse sélectionnée à partir du groupe constitué des cellules d'une leucémie, d'un lymphome, d'un cancer neurologique, d'un mélanome, d'un cancer du sein, d'un cancer des poumons, d'un cancer des voies aéro-digestives supérieures, d'un cancer des voies gastro-intestinales, d'un cancer du foie, d'un cancer du pancréas, d'un cancer des voies génito-urinaires, d'un cancer de la prostate, d'un cancer des cellules rénales, d'un cancer des os et d'un cancer des voies vasculaires.
  5. Antigène et anticorps selon la revendication 1, où l'antigène est une molécule produite par un micro-organisme infectieux.
  6. Antigène et anticorps selon la revendication 5, où le micro-organisme infectieux est un virus.
  7. Antigène et anticorps selon la revendication 6, où le virus est un rétrovirus.
  8. Antigène et anticorps selon la revendication 5, où le micro-organisme infectieux est sélectionné à partir du groupe constitué d'une bactérie, d'un champignon et d'un protozoaire parasite.
  9. Antigène et anticorps selon la revendication 1, où le sujet est un être humain.
  10. Antigène et anticorps selon la revendication 1, où l'antigène est une cellule dendritique comprenant une molécule de complexe majeur d'histocompatibilité (CMH) ayant un épitope à peptide lié à celle-ci, où l'épitope à peptide est un fragment d'un TAA ou un fragment d'un polypeptide produit par un micro-organisme infectieux.
  11. Antigène et anticorps selon la revendication 10, où la molécule de CMH est une molécule de CMH de classe I.
  12. Antigène et anticorps selon la revendication 10, où la molécule de CMH est une molécule de CMH de classe II.
  13. Antigène et anticorps selon la revendication 1, où la réponse immunitaire est une réponse d'une cellule T.
  14. Antigène et anticorps selon la revendication 13, où la cellule T est une cellule T CD8+.
  15. Antigène et anticorps selon la revendication 13, où la cellule T est une cellule T CD4+.
  16. Antigène et anticorps selon la revendication 1, où l'antigène est une cellule hybride.
  17. Antigène et anticorps selon la revendication 16, où la cellule hybride est un produit de fusion d'une cellule tumorale et d'une cellule dendritique.
  18. Antigène et anticorps selon la revendication 1, où l'antigène est une cellule tumorale, un lysat de cellule tumorale, un TAA, un épitope à peptide d'un TAA, ou une protéine de choc thermique liée à un épitope à peptide de protéine exprimé par une cellule tumorale.
  19. Antigène et anticorps selon la revendication 1, où l'antigène est une cellule dendritique qui a été incubée avec des cellules tumorales, un lysat de cellule tumorale, un TAA, un épitope à peptide d'un TAA, ou une protéine de choc thermique liée à un épitope à peptide exprimé par une cellule tumorale.
  20. Antigène et anticorps selon la revendication 18, où la cellule tumorale est transfectée ou transformée avec un acide nucléique codant une cytokine ou un facteur de croissance.
  21. Antigène et anticorps selon la revendication 20, où la cytokine est un facteur stimulant la formation et le développement de colonie de macrophages et de granulocytes (GM-CSF).
  22. (a) Antigène pour lequel un récepteur de cellule T est spécifique et anticorps agoniste liant 4-1BB,
    (b) acide nucléique codant l'antigène et l'anticorps agoniste liant 4-1BB,
    (c) antigène et acide nucléique codant l'anticorps agoniste liant 4-1BB,
    ou
    (d) acide nucléique codant l'antigène et acide nucléique codant l'anticorps agoniste liant 4-1BB,
    destinés à une utilisation sous la forme d'un médicament ou d'une combinaison de médicaments afin de prévenir l'induction d'anergie ou d'inverser une anergie dans une cellule T chez un sujet.
  23. Acide nucléique codant l'antigène et acide nucléique codant l'anticorps agoniste liant 4-1BB selon la revendication 22, où l'acide nucléique codant l'antigène et l'acide nucléique codant l'anticorps liant 4-1BB sont la même molécule d'acide nucléique.
  24. Acide nucléique codant l'antigène et acide nucléique codant l'anticorps agoniste liant 4-1BB selon la revendication 22, où l'acide nucléique codant l'antigène ou l'anticorps liant 4-1BB est transfecté ou transduit dans une cellule, où la cellule est une cellule, ou une descendance d'une cellule qui, avant la transfection ou la transduction, a été obtenue à partir d'un mammifère.
  25. Antigène et anticorps selon l'une quelconque des revendications précédentes, où l'anticorps est un fragment de liaison d'antigène, en particulier Fab, F(ab')2, Fv ou un fragment de Fv à une seule chaîne.
  26. Utilisation
    (a) d'un antigène pour lequel un récepteur de cellule T est spécifique, et
    (b) d'un anticorps agoniste liant 4-1BB,
    pour la fabrication d'un médicament ou d'une combinaison de médicaments afin de générer une réponse de cellule T améliorée chez un sujet.
  27. Utilisation selon la revendication 26, dans laquelle l'antigène est (a) un antigène associé à une tumeur (TAA) ou (b) un fragment fonctionnel d'un TAA.
  28. Utilisation selon la revendication 26, dans laquelle l'antigène est un polypeptide.
  29. Utilisation selon la revendication 27, dans laquelle le TAA est une molécule produite par une cellule cancéreuse sélectionnée à partir du groupe constitué des cellules d'une leucémie, d'un lymphome, d'un cancer neurologique, d'un mélanome, d'un cancer du sein, d'un cancer des poumons, d'un cancer des voies aéro-digestives supérieures, d'un cancer des voies gastro-intestinales, d'un cancer du foie, d'un cancer du pancréas, d'un cancer des voies génito-urinaires, d'un cancer de la prostate, d'un cancer des cellules rénales, d'un cancer des os et d'un cancer des voies vasculaires.
  30. Utilisation selon la revendication 26, dans laquelle l'antigène est une molécule produite par un micro-organisme infectieux.
  31. Utilisation selon la revendication 30, dans laquelle le micro-organisme infectieux est un virus.
  32. Utilisation selon la revendication 31, dans laquelle le virus est un rétrovirus.
  33. Utilisation selon la revendication 30, dans laquelle le micro-organisme infectieux est sélectionné à partir du groupe constitué d'une bactérie, d'un champignon et d'un protozoaire parasite.
  34. Utilisation selon la revendication 26, dans laquelle le sujet est un être humain.
  35. Utilisation selon la revendication 26, dans laquelle l'antigène est une cellule dendritique comprenant une molécule de complexe majeur d'histocompatibilité (CMH) ayant un épitope à peptide lié à celle-ci, où l'épitope à peptide est un fragment d'un TAA ou un fragment d'un polypeptide produit par un micro-organisme infectieux.
  36. Utilisation selon la revendication 35, dans laquelle la molécule de CMH est une molécule de CMH de classe I.
  37. Utilisation selon la revendication 35, dans laquelle la molécule de CMH est une molécule de CMH de classe II.
  38. Utilisation selon la revendication 26, dans laquelle la cellule T est une cellule T CD8+.
  39. Utilisation selon la revendication 26, dans laquelle la cellule T est une cellule T CD4+.
  40. Utilisation selon la revendication 26, dans laquelle l'antigène est une cellule hybride.
  41. Utilisation selon la revendication 40, dans laquelle la cellule hybride est un produit de fusion d'une cellule tumorale et d'une cellule dendritique.
  42. Utilisation selon la revendication 26, dans laquelle l'antigène est une cellule tumorale, un lysat de cellule tumorale, un TAA, un épitope à peptide d'un TAA, ou une protéine de choc thermique liée à un épitope à peptide de protéine exprimé par une cellule tumorale.
  43. Utilisation selon la revendication 26, dans laquelle l'antigène est une cellule dendritique qui a été incubée avec des cellules tumorales, un lysat de cellule tumorale, un TAA, un épitope à peptide d'un TAA, ou une protéine de choc thermique liée à un épitope à peptide exprimé par une cellule tumorale.
  44. Utilisation selon la revendication 42, dans laquelle la cellule tumorale est transfectée ou transformée avec un acide nucléique codant une cytokine ou un facteur de croissance.
  45. Utilisation selon la revendication 44, dans laquelle la cytokine est un facteur stimulant la formation et le développement de colonie de macrophages et de granulocytes (GM-CSF).
  46. Utilisation
    (a) d'un antigène pour lequel un récepteur de cellule T est spécifique et d'un anticorps agoniste liant 4-1BB,
    (b) d'un acide nucléique codant l'antigène et l'anticorps agoniste liant 4-1BB,
    (c) de l'antigène et d'un acide nucléique codant l'anticorps agoniste liant 4-1BB,
    ou
    (d) d'un acide nucléique codant l'antigène et d'un acide nucléique codant l'anticorps agoniste liant 4-1BB,
    pour la fabrication d'un médicament ou d'une combinaison de médicaments afin de prévenir l'induction d'anergie ou d'inverser une anergie dans une cellule T chez un sujet.
  47. Utilisation selon la revendication 46, dans laquelle l'acide nucléique codant l'antigène et l'acide nucléique codant l'anticorps liant 4-1BB sont la même molécule d'acide nucléique.
  48. Utilisation selon la revendication 46, dans laquelle l'acide nucléique codant l'antigène ou l'anticorps liant 4-1BB est transfecté ou transduit dans une cellule, où la cellule est une cellule, ou une descendance d'une cellule qui, avant la transfection ou la transduction, a été obtenue à partir d'un mammifère.
  49. Utilisation selon l'une quelconque des revendications 26 à 48, dans laquelle l'anticorps est un fragment de liaison d'antigène, en particulier Fab, F(ab')2, Fv ou un fragment de Fv à une seule chaîne.
  50. Procédé in vitro destiné à activer une cellule T, le procédé comprenant la mise en culture d'un échantillon de cellule comprenant une cellule T avec un antigène pour lequel un récepteur de cellule T est spécifique et un anticorps agoniste liant 4-1BB.
  51. Procédé in vitro destiné à prévenir l'induction d'anergie ou d'inverser une anergie dans une cellule, le procédé comprenant la mise en contact de la cellule T avec :
    (a) un antigène pour lequel un récepteur de cellule T est spécifique, et
    (b) un anticorps agoniste liant 4-1BB.
  52. Procédé selon la revendication 51, dans lequel l'antigène est (a) un antigène associé à une tumeur (TAA) ou (b) un fragment fonctionnel d'un TAA.
  53. Procédé selon la revendication 51, dans lequel l'antigène est un polypeptide.
  54. Procédé selon la revendication 52, dans lequel le TAA est une molécule produite par une cellule cancéreuse sélectionnée à partir du groupe constitué des cellules d'une leucémie, d'un lymphome, d'un cancer neurologique, d'un mélanome, d'un cancer du sein, d'un cancer des poumons, d'un cancer des voies aéro-digestives supérieures, d'un cancer des voies gastro-intestinales, d'un cancer du foie, d'un cancer du pancréas, d'un cancer des voies génito-urinaires, d'un cancer de la prostate, d'un cancer des cellules rénales, d'un cancer des os et d'un cancer des voies vasculaires.
  55. Procédé selon la revendication 51, dans lequel l'antigène est une molécule produite par un micro-organisme infectieux.
  56. Procédé selon la revendication 55, dans lequel le micro-organisme infectieux est un virus.
  57. Procédé selon la revendication 56, dans lequel le virus est un rétrovirus.
  58. Procédé selon la revendication 55, dans lequel le micro-organisme infectieux est sélectionné à partir du groupe constitué d'une bactérie, d'un champignon et d'un protozoaire parasite.
  59. Procédé selon la revendication 51, où le procédé comprend la mise en contact de la cellule T avec une cellule dendritique comprenant une molécule de complexe majeur d'histocompatibilité (CMH) ayant un épitope à peptide lié à celle-ci, où l'épitope à peptide est un fragment d'un TAA ou un fragment d'un polypeptide produit par un micro-organisme infectieux.
  60. Procédé selon la revendication 59, dans lequel la molécule de CMH est une molécule de CMH de classe I.
  61. Procédé selon la revendication 59, dans lequel la molécule de CMH est une molécule de CMH de classe II.
  62. Procédé selon la revendication 51, dans lequel la cellule T est une cellule T CD8+.
  63. Procédé selon la revendication 51, dans lequel la cellule T est une cellule T CD4+.
  64. Procédé selon la revendication 51, où le procédé comprend la mise en contact de la cellule T avec une cellule hybride.
  65. Procédé selon la revendication 64, dans lequel la cellule hybride est un produit de fusion d'une cellule tumorale et d'une cellule dendritique.
  66. Procédé selon la revendication 51, où le procédé comprend la mise en contact de la cellule T avec une cellule tumorale, un lysat de cellule tumorale, un TAA, un épitope à peptide d'un TAA, ou une protéine de choc thermique liée à un épitope à peptide de protéine exprimé par une cellule tumorale.
  67. Procédé selon la revendication 51, où le procédé comprend la mise en contact de la cellule T avec une cellule dendritique qui a été incubée avec des cellules tumorales, un lysat de cellule tumorale, un TAA, un épitope à peptide d'un TAA, ou une protéine de choc thermique liée à un épitope à peptide exprimé par une cellule tumorale.
  68. Procédé selon la revendication 66, dans lequel la cellule tumorale est transfectée ou transformée avec un acide nucléique codant une cytokine ou un facteur de croissance.
  69. Procédé selon la revendication 67, dans lequel la cytokine est un facteur stimulant la formation et le développement de colonie de macrophages et de granulocytes (GM-CSF).
  70. Procédé selon l'une quelconque des revendications 50 à 69, dans lequel l'anticorps est un fragment de liaison d'antigène, en particulier Fab, F(ab')2, Fv ou un fragment de Fv à une seule chaîne.
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US7651686B2 (en) 2010-01-26
US8163550B2 (en) 2012-04-24
ATE435655T1 (de) 2009-07-15
EP1434596A1 (fr) 2004-07-07
US20050013811A1 (en) 2005-01-20
US20100266611A1 (en) 2010-10-21
WO2003049755A1 (fr) 2003-06-19
ES2328025T3 (es) 2009-11-06
US8772026B2 (en) 2014-07-08
AU2002364935A1 (en) 2003-06-23
DE60232895D1 (de) 2009-08-20

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